微小RNA-20b-5p通过靶向NFKBIZ基因调控急性肺损伤中巨噬细胞极化和铁死亡

MicroRNA-20b-5p regulates macrophage polarization and ferroptosis in acute lung injury by targeting the NFKBIZ gene

目的探讨微小RNA(miR)-20b-5p在急性肺损伤中调控巨噬细胞极化与铁死亡的具体作用机制。方法以脂多糖和干扰素-γ(IFN-γ)诱导RAW264.7细胞后, 采用免疫印迹法和流式细胞术检测miR-20b-5p、miR-20b-5p抑制剂和NFKBIZ (NFKB inhibitor Zeta)对巨噬细胞极化指标诱导型一氧化氮合酶(iNOS)与CD86表达的影响。通过双荧光素酶报告基因检测证实miR-20b-5p与NFKBIZ之间的相互调控关系。用流式细胞仪和免疫印迹法分别检测miR-20b-5p对巨噬细胞内活性氧(ROS)和铁死亡相关基因表达的调控。构建急性肺损伤动物模型, 研究miR-20b-5p对肺组织损伤的保护作用。两组间差异比较采用t检验, 3组或以上的组间差异采用方差分析。结果经脂多糖和IFN-γ处理6、12、18、24 h后, RAW264.7细胞向M1极化(NC比miR-20b-5p:26.00±2.65比52.67±2.52, t=12.650, P<0.001), miR-20b-5p表达水平低于NC组[0 h比6 h:(101.00±3.61)%比(42.65±1.61)%、0 h比12 h:(101.00±3.61)%比(44.93±1.29)%、0 h比18 h:(101.00±3.61)%比(56.10±1.09)%、0 h比24 h:(101.00±3.61)%比(51.94±0.42)%, 0 h比6 h:t=25.600, 0 h比12 h:t=25.36, 0 h比18 h:t=20.65, 0 h比24 h:t=23.41, 均P<0.001]。miR-20b-5p的过表达能抑制巨噬细胞iNOS和CD86蛋白表达水平(iNOS:97.33±1.53比22.77±4.14、CD86:101.67±2.52比33.30±6.00, iNOS:t=23.230, CD86:t=21.300, 均P<0.001), 下调miR-20b-5p的表达则上调巨噬细胞iNOS和CD86蛋白表达水平(iNOS:97.33±1.53比344.67±6.51、CD86:101.67±2.52比214.00±7.55, iNOS:t=58.300, CD86:t=26.480, 均P<0.001)。免疫印迹法和双荧光素酶实验的结果表明miR-20b-5p是通过直接靶向NFKBIZ基因发挥上述作用。此外, miR-20b-5p的过表达显著抑制LPS诱导的ROS水平(37.60±1.82比17.50±0.97, F=180.400, P<0.0001), 并上调NRF2和GPX4蛋白表达水平(NRF2:101.00±7.22比277.19±13.49、GPX4:100.00±5.98比178.24±8.99, NRF2:F=375.900, GPX4:F=109.300, 均P<0.001)。通过对急性肺损伤动物模型的研究, miR-20b-5p显著抑制肺组织损伤, 肺损伤指标显著下调(3.45±0.18比1.25±0.06, t=20.080, P<0.001)。结论 miR-20b-5p可通过靶向NFKBIZ抑制巨噬细胞的极化和铁死亡。

Objective To clear up the role of microRNA(miR)-20b-5p in controlling ferroptosis and macrophage polarization during acute lung injury(ALI).Methods After stimulating RAW264.7 cells with lipopolysaccharide(LPS)and interferon-γ(IFN-γ),Western blotting and flow cytometry assays were used to assess the effects of miR-20b-5p,miR-20b-5p inhibitor,and NFKB inhibitor Zeta(NFKBIZ)on the expression of macrophage polarization markers[inducible nitric oxide synthase(iNOS)and CD86].The association of miR-20b-5p and NFKBIZ was validated using a dual-luciferase reporter gene assay.Flow cytometry and Western blotting assays were used to determine the modulatory effects of miR-20b-5p on reactive oxygen species(ROS)and ferroptosis-related gene expression in macrophages,respectively.To investigate the protective effect of miR-20b-5p on sepsis-induced lung tissue injury,an animal model of ALI was developed.Results After treatment with LPS and IFN-γfor 6,12,18 and 24 h,RAW264.7 cells polarized towards M1[NC vs.miR-20b-5p:(26.00±2.65)cells/field vs.(52.67±2.52)cells/field,t=12.650,P<0.001]and the expression level of miR-20b-5p significantly decreased[0 h vs.6 h:(101.00±3.61)%vs.(42.65±1.61)%,0 h vs.12 h:(101.00±3.61)%vs.(44.93±1.29)%,0 h vs.18 h:(101.00±3.61)%vs.(56.10±1.09)%,0 h vs.24 h:(101.00±3.61)%vs.(51.94±0.42)%,0 h vs.6 h:t=25.600,0 h vs.12 h:t=25.36,0 h vs.18 h:t=20.65,0 h vs.24 h:t=23.41,all P<0.001].Overexpression of miR-20b-5p could inhibit the expression of iNOS and CD86 in macrophages(iNOS:97.33±1.53 vs.344.67±6.51,CD86:101.67±2.52 vs.214.00±7.55,iNOS:t=23.230,CD86:t=21.300,both P<0.001),while downregulation of miR-20b-5p could upregulate the expression of iNOS and CD86 in macrophages(iNOS:97.33±1.53 vs.344.67±6.51,CD86:101.67±2.52 vs.214.00±7.55,iNOS:t=58.300,CD86:t=26.480,both P<0.001).The results of Western blotting and dual-Luciferase experiments showed that miR-20b-5p could directly target NFKBIZ gene.In addition,overexpression of miR-20b-5p significantly inhibited ROS levels(37.60±1.82 vs.17.50±0.97,F=180.400,P<0.0001)and upregulated the expression of NRF2 and GPX4(NRF2:101.00±7.22 vs.277.19±13.49,GPX4:100.00±5.98 vs.178.24±8.99,NRF2:F=375.900,GPX4:F=109.300,both P<0.001).Through research on animal models of ALI,miR-20b-5p significantly inhibited lung tissue injury and significantly downregulated lung injury indicators(3.45±0.18 vs.1.25±0.06,t=20.080,P<0.001).Conclusion MiR-20b-5p can inhibit macrophage polarization and ferroptosis by targeting NFKBIZ.